1. Field of the Invention
The present invention relates to methods of identifying drug-resistant and/or drug-sensitive cells, for example, breast cancer and brain tumor cells, on the basis of different ion and/or second messenger dynamics between a drug-sensitive and drug-resistant cell. For example, the invention provides measuring the comparative decay rates of a cellular ion, such as calcium, released into the intracellular compartment of drug sensitive and/or drug resistant cells. The present invention also provides methods for screening compounds that modulate the ionic, dynamics of a cell as well as methods of determining drug resistance/sensitivity of cancer cells from cancer patients and/or designing cancer therapy based on of the ionic dynamics of cancer cells from a particular patient.
2. Discussion of the Background
The emergence of drug-resistant cancer cells represents a major therapeutic problem. When a cancer develops drug resistance, treatment protocols must be modified, for example, by administering a higher drug dose or switching the patient to a different drug combination or regimen.
Cancer cells that are exposed to a cytotoxic agent for a prolonged period of time often become resistant to that agent, as well as other chemically unrelated compounds. This resistance represents a major cause for chemotherapy failure in cancer patients, such as breast cancer patients. Moreover, some cancers, such as breast cancer, may develop resistance to multiple drugs further aggravating the problem. Such drug-resistances may be attributed to both phenotypic and genotypic changes in a drug-resistant cancer cell. For example, the expression or upregulation of P-glycoprotein (P-gp, permeability glycoprotein) or other multidrug resistance genes, can alter the absorption, distribution, or clearance of a variety of compounds. Drug-resistant human cancer cells are well known and can be produced by exposing a drug-sensitive parental cell line to a drug, and then selecting drug resistant variants of the parental cell line. Such cells are valuable research tools for studying the mechanisms associated with the development of drug resistance. In vitro, drug-resistant (DR) cells and drug-sensitive (DS) cells exhibit a variety of different biochemical features. For example, compared to drug-sensitive breast cancer cells, drug resistant breast cancer cells may exhibit one or more of the following biochemical features or phenotypes: over-expression of ATP-driven membrane drug efflux pumps, such as P-gp, MRP (Barrand, M. A. et al., Gen. Pharmacol. 28:639-45, 1997) and BCRP (Doyle, L. A. et al., Proc. Natl. Acad. Sci., U.S.A., 26:15665-70, 1998); over-expression of nucleoside transporters; reduced susceptibility to oxygen radicals; increased accumulation of glycolipids; resistance to apoptosis or programmed cell death; highly acidified organelles and elevated cytosolic pH; increased protein kinase Cα content; lower basal cyclic AMP levels; or alterations in enzyme activity or gene expression (Barrand et al Gen Pharmacol. 28: 63945, 1997; Doyle et al Proc. Natl. Acad. Sci. 26.-15665-15670, 1998; Morgan et al Cancer Chemother Pharmacol 29: 127-32, 1991; Sinha et al Biochemistry 26:3776-81, 1987; Lavie et al J Biol Chem 272: 1682-7, 1997; Ogretmen et al Int J Cancer 67:608-14, 1996; Altan et al J Exp Med 187:1583-98, 1998; Blobe et al J Biol Chem. 268:658-64, 1993; Mestdagh et al Biochem Phannacol 48:709-16, 1994; Chen et al Biochem Pharmacol. 49:1691-701, 1995; Wosikowski et al Clin Cancer Res. 3:2405-14, 1997; Schneider et al Cancer Res 54:152-8, 1994).
However, standard methods for determining whether a cancer cell has acquired drug resistance are time-consuming, lack specificity and sensitivity, require the processing of large numbers of cells, and result in the destruction of the tested cells. These methods conventionally involve exposing cells cultured in vitro to progressively increasing concentrations of a drug. Drug-resistant cells are then identified by the ability to survive or proliferate in a particular concentration of drug. However, these assays generally require the processing of large cell populations to establish drug resistance, which is measured by parameters such as IC50, the dose of the drug that kills 50% of the cell population. Additionally, the cells must be exposed to the drug for a period of 24 to 48 hours and in some assays greater than 48 hours.
Moreover, the results of such methods may lack specificity as a cultured cell may be phenotypically or genotypically altered by prolonged exposure to an in vitro culture medium (Rubin (1990) Cancer and Metastasis Reviews 9:1-20; Wolffe and Tata (1984) FEBS Letters 176(1):8-15). Moreover, the relative percentages of drug-sensitive and drug-resistant cells are difficult to determine when cells are cultured in vitro, due to different proliferation rates of drug sensitive and drug resistant cells. Similarly, when a culture contains cancer cells with different degrees of drug resistance or different proliferative abilities, conventional methods may lead to a loss of that fraction of the culture, which is of particular importance. For example, while conventional methods would yield an IC50 value for an entire population of cultured cells, they would not necessarily discriminate between the fraction of a cell population that represents rapidly growing drug resistant cells, and the rest of the population that may be slower growing, all of which are less drug resistant and/or drug sensitive cells.
The constraints imposed by the standard methods emphasize the need for faster, more specific, and sensitive assays that can be performed with fewer cells, preferentially at the single cell level, and which permit facile recovery of the tested cells for further expansion or testing.
The role of ion dynamics for distinguishing between drug resistant and drug-sensitive cells has not been previously investigated. For example, calcium is one of the most ubiquitous second messengers involved in a wide variety of cellular responses. The maintenance of physiological calcium levels within a cell, as well as the functional elevation of calcium levels, are highly regulated processes. Consequently, either an impaired or an excessive response of a cell to a calcium-evoking signal may negatively impact cell survival.
Thus, prior efforts have focused on the development of new drugs that might modulate cellular calcium dynamics; for example, by promoting an intracellular increase of calcium, when the cell itself is impaired in its ability to respond to calcium-elevating stimuli; restoring calcium homeostasis, when the cell is unable to reduce sustained intracellular calcium increase upon stimulation; or by inhibiting calcium increase, when a calcium response is undesirable, such as in pathophysiological states.
However, very little is known about the potential role played by cellular ions, and in particular, calcium ions, in the development of drug resistance in cancer cells. One report shows that in the adriamycin-resistant MCF-7 cell line of human breast cancer, resting Ca2+1 levels are higher than in parental, drug-sensitive cells and that resting Ca2+1 levels were reduced by verapamil, a calcium channel blocker and P-gp modulator (Mestdagh, N. et al. Biochem. Pharmacol. 48:709-716 (1994)). Adriamycin-resistant MCF-7 cells also are known to overexpress the epidermal growth factor (EGF) receptor, whose activation leads to increased intracellular calcium levels ([Ca2+]1). The reversion of such cells to drug-sensitivity correlated with the return of EGF levels to the levels exhibited by the drug-sensitive parental MCF-7 cells (Dickstein, B. et al. J Cell Physiol 157:110-118, (1993)). In leukemia cells, calcium levels have been reported to modulate cell sensitivity to drugs (Adwankar, M K & Chitnis, M P Neoplasma 37:31-36, (1990)); and in ovarian cancer cells, differences in calcium handling have also been reported between DS and DR cells (McAlroy, H. L. et al. Exp. Physiol. 84:285-97, (1999)).
Empirically, it has been found that compounds that independently modulate Ca2+1 dynamics can reverse drug resistance or chemosensitize a number of different cancers. For example, verapamil, a blocker of voltage-operated Ca2+ channels, and cyclosporin A, an inhibitor of calcineurin, which is a Ca2+/calmodulin-dependent protein phosphatase, are among the most widely studied chemosensitizers for tumors overexpressing P-gp.
Similarly, chemical modulators of MRP (multidrug resistance-associated protein), such as probenecid and genistein, are known. Probenecid is an organic anion transport blocker and can either depress or increase Ca2+1 responses in different cell systems at the concentrations used for chemosensitization. Genistein is a protein tyrosine kinase inhibitor and can prevent capacitative Ca2+ entry upon agonist-evoked Ca2+1 release. However, the importance of cellular ionic dynamics, such as calcium levels, calcium flux, and/or intracellular calcium kinetics for distinguishing drug-resistant and drug-sensitive cancer cells has not been previously described.
In view of the high morbidity and mortality associated with the development of drug-resistance in cancer cells, there is a pronounced need to develop rapid methods for diagnosing the presence, type and frequency of drug-resistant cancer cells, as well as diagnosing and appropriately treating subjects having drug resistant cancer. Moreover, new methods are needed for screening compounds that modulate or reverse drug-resistance in such cells.